This invention relates to a newly identified receptor, the mouse growth hormone secretagogue receptor (mGHS-R), nucleic acids encoding this receptor; and to the use of a mGHS-R to identify growth hormone secretagogues and compounds that modulate mGHS-R function.
Growth hormone secretagogues (GHSs) and secretagogue-like compounds, both peptide and non-peptide, bind to and exert their biological effects (i.e., release of growth hormone (GH)) through a G protein-coupled receptor (GPC-R) distinct from the receptors for growth hormone releasing hormone (GHRH) and somatostatin (SST) (Pong et al., 1996 Mol. Endocrin. 10:57-61). The molecular cloning of this growth hormone secretagogue receptor (GHS-R) capitalized on the pivotal observation that GHSs transduce their signal through activation of the phospholipase C pathway (Cheng et al., 1991 Endocrinology 129:3337-3342; Howard et al., 1996 Science 273:974-977). cDNA and genomic DNA cloning from human, swine, and rat showed that the GHS-R is a protein of 364/366 amino acids containing 7 putative alpha-helical transmembrane (TM) domains, a signature feature of GPC-Rs (Howard et al. 1996; McKee et al., 1997 Mol. Endocrin. 11:415-423). In all species evaluated, the GHS-R is encoded by a single highly-conserved gene containing one intron, placed at the C-terminal end of TM domain 5.
The biology of the growth hormone secretagogues (GHSs) is still in a relatively early stage of development. Research is focused on identification of the GHS natural ligand system and understanding the role of the GHS-R in brain regions (substantia nigra, dentate gyrus, hippocampus) other than those traditionally thought to be involved in GH secretion (Bennett et al. 1997; Guan et al. 1997).
It would be desirable to know the molecular structure of growth hormone secretagogue receptors in order to analyze this new receptor family and understand its normal physiological role in: concert with the actions of GHRH and somatostatin. This could lead to a better understanding of the in vivo processes which occur upon ligand-receptor binding. Further, it would be desirable to use cloned-growth hormone secretagogue receptors as essential components of an assay system which can identify new growth hormone secretagogues which would confer a significant benefit on children and adults deficient in growth hormone, the frail elderly, those in post-hip fracture rehabilitation and post-operative recovery patients.
This invention relates to a novel receptor, mouse growth hormone secretagogue receptor (mGHS-R), which is free from receptor associated proteins. A further aspect of this invention is mGHS-R which is isolated or purified.
Another aspect of this invention is mGHS-Rs which are encoded by substantially the same nucleic acid sequence, but which have undergone changes in splicing or other RNA processing-derived modifications or mutagenesis induced changes, so that the expressed protein has a homologous, but different amino acid sequence from the native form. These variant forms may have different and/or additional functions in animal physiology or in vitro in cell based assays.
Growth hormone secretagogue receptors are proteins containing various functional domains, including one or more domains which anchor the receptor in the cell membrane, and at least one ligand binding domain. As with many receptor proteins, it is possible to modify many of the amino acids, particularly those which are not found in the ligand binding domain, and still retain at least a percentage of the biological activity of the original receptor. Thus, this invention specifically includes modified functionally equivalent mGHS-Rs which have deleted, truncated, or mutated N-terminal portions. This invention also specifically includes modified functionally equivalent mGHS-Rs which contain modified and/or deletions in other domains, which are not accompanied by a loss of functional activity.
Additionally, it is possible to modify other functional domains such as those that interact with second messenger effector systems, by altering binding specificity and/or selectivity. Such functionally equivalent mutant receptors are also within the scope of this invention.
A further aspect of this invention are nucleic acids which encode a mouse growth hormone secretagogue receptor or a functional equivalent. These nucleic acids may be free from associated nucleic acids, or they may be isolated or purified. For most cloning purposes, cDNA is a preferred nucleic acid, but this invention specifically includes other forms of DNA as well as RNAs which encode a mGHS-R or a functional equivalent.
Yet another aspect of this invention relates to vectors which comprise nucleic acids encoding mGHS-R or a functional equivalent. These vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial chromosomes, transposable elements and other forms of episomal or integrated DNA that can encode a mGHS-R. It is well within the skill of the ordinary artisan to determine an appropriate vector for a particular gene transfer or other use.
A further aspect of this invention are host cells which are transformed with a vector comprising a gene which encodes a mouse growth hormone secretagogue receptor or a functional equivalent. The host cell may or may not naturally express a GHS-R on the cell membrane. Preferably, once transformed, the host cells are able to express the mouse growth hormone secretagogue receptor or a functional equivalent on the cell membrane. Depending on the host cell, it may be desirable to adapt the DNA so that particular codons are used in order to optimize expression. Such adaptations are known in the art, and these nucleic acids are also included within the scope of this invention. Generally, mammalian cell lines, such as COS, HEK-293, CHO, HeLa, NS/0, CV-1, GC, GH3 or VERO cells are preferred host cells, but other cells and cell lines such as Xenopus oocytes or insect cells, may also be used.
Another aspect of this invention is a process for identifying nucleic acids encoding mouse growth hormone secretagogue related receptors comprising hybridizing a first nucleic acid encoding a mouse growth hormone secretagogue receptor with a second nucleic acid suspected of comprising nucleic acids encoding a growth hormone secretagogue receptor, wherein the hybridizing takes place under relaxed or moderate post hybridizational washing conditions; and identify areas of the second nucleic acid where hybridization occurred.